Worldwide eradication of smallpox (a disease resulting from infection with variola virus) by vaccination with vaccinia virus (VV) was an outstanding success. Vaccination was discontinued in the U.S. in 1972 and therefore the entire population must be considered susceptible to smallpox. In response to a smallpox attack by bioterrorists, live VV vaccination is the only licensed prophylaxis (Breman et al., 1998, N. Eng. J. Med. 339:556-559; Franz et al., 1997, JAMA 278:399-411, Henderson, 1999, Science. 283:1279-82; Henderson et al., 2001, Clin. Infect. Dis. 33:1057-1059). However, mass immunization is limited by the fact that the live-virus vaccine has complications, especially in immunocompromised hosts, pregnant women and infants. An attenuated form of the vaccine, called MVA (modified VV Ankara), is an option for immunizing such at risk populations (Meyer et al., 1991, J. Gen. Virol. 72: 1031-1038; Moss, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:11341-1134). However, this virus has not been tested to determine whether it can prevent smallpox and production of the virus in chicken embryo cells can be problematic.
Viruses in the Poxyiridae family, including VV and variola virus, are characterized by a large linear double-stranded DNA genome (130-300 kb) packaged in a relatively large particle, and a cytoplasmic site of replication (reviewed by Moss, 1996, In: Fields Virology, Knipe et al. eds., vol. 3, pp. 2637-2671, Lippincott-Raven, Philadelphia). Two distinct types of infectious particles are produced during poxvirus replication: the intracellular mature virions (IMV) and the extracellular enveloped virions (EEV) (Smith et al., 1998, Adv. Exp. Med. Biol. 440:395-414; Sodeik et al., 2002, Trends Microbiol. 10:15-24). A third form, CEV (cell-associated enveloped virus) is very similar in composition to EEV but remains tightly associated with the membrane of the cell it exited from.
IMV is the most abundant form of infectious particle and is surrounded by a lipid bilayer studded with membrane proteins, including L1R, A27L, A17L, D8L and H3L. EEVs consist of IMVs wrapped by a double membrane acquired from the Golgi apparatus (Hiller et al., 1985, J. Virol. 55:651-659; Moss, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:1131-1134). The EEV membrane is studded with EEV-specific proteins, for example A33R and B5R. Released EEVs are responsible for widespread dissemination of VV in vivo and form the distinct comet-shaped plaques of VV seen in vitro (Boulter, 1969, Proc. R. Soc. Med. 62:295-7; Boulter et al., 1973, Prog. Med. Virol. 16:86-108; Law et al., 2002, J. Gen. Virol. 83:209-222; Payne, 1979, J. Virol. 31:147-155; Payne, 1980, J. Gen. Virol. 50:89-100).
IMV and EEV are equally infectious in vitro. The consensus is that IMVs enter cells via direct fusion with the plasma membrane. The entry of EEV is controversial. In one study, both EEV and IMV entered at neutral pH, and entry of both forms was insensitive to lysosomatropic agents, implying direct fusion (Doms et al., 1990, J. Virol. 64:4884-4892). Recent evidence indicates that cytochalasin D, an actin-depolymerizing drug, affected the entry of both forms and that entry of EEV, but not IMV, was inhibited by chloroquine and ammonium chloride (Ichihashi et al., 1996, Virology. 217:478-485; Vanderplasschen et al., 1998, J. Gen. Virol. 79:877-887). This suggests that EEVs gain entry to cells via an endocytic pathway. These differences in entry may reflect differences in receptor usage by as yet unidentified virion proteins that are unique to each form.
IMV is thought to be responsible for VV spread between hosts because the membrane of EEV is fragile (Ichihashi et al., 1996, Virology. 217:478-485). EEV is postulated to be responsible for widespread dissemination of virus within the host (Boulter et al., 1969, Proc. R. Soc. Med. 62:295-7; Boulter et al., 1973, Prog. Med. Virol. 16:86-108; Payne et al., 1980, J. Gen. Virol. 50:89-100). In support of this, a correlation was found between the virulence of VV strains and their ability to form EEV in vitro (Payne et al., 1980, J. Gen. Virol. 50:89-100). Earlier studies showed that immunity to EEV conferred protection against poxvirus infections (Appleyard et al., 1974, J. Gen. Virol. 23:197-200; Appleyard et al., 1971, J. Gen. Virol. 13:9-17; Boulter et al., 1973, Prog. Med. Virol. 16:86-108; Payne et al., 1980, J. Gen. Virol. 50:89-100; Turner et al., 1971, J. Gen. Virol. 13:19-25). In support of this, two EEV proteins, A33R and B5R (Galmiche et al., 1999, Virology 254:71-80), or DNA encoding them (Hooper et al., 2000, Virology 266:329-339) protected mice from VV challenge. However, IMV proteins are also likely to play an important role in protection. For example, vaccination with the DNA encoding the full-length IMV protein L1R protected mice from VV challenge (Hooper et al., 2000, Virology. 266:329-39).
In the event of bioterrorism using smallpox, the only commercially approved smallpox vaccine available in the U.S. is Wyeth Dryvax, which consists of lyophilized VV prepared from calves. This vaccine caused adverse events in vaccinees (Kempe, 1960, 26:176-189; Lane et al. JAMA 212:441-4; Lane et al., 1969, New Eng. J. Med. 281:1201-1208; Lane et al., 1970, J. Infect. Dis. 122:303-952-54), ranging from mild local reactions to serious sequelae, such as postvaccinal encephalopathy, encephalitis and death. The vaccine is contraindicated in immunosuppressed patients, pregnant women, and infants. In a 1968 surveillance study of people receiving the vaccine for the first time, there were approximately 75 complications per million vaccinations with an overall death rate of one per million (Lane et al., 1969, New Eng. J. Med. 281:1201-1208). However, since complications from smallpox vaccination were not reportable, these numbers underestimate the actual rates.
The only approved treatment available in the U.S. for vaccine related complications is vaccinia immune globulin (VIG), administered intramuscularly at about 0.6 ml/kg as described in Rosenthal et al. (2001, Emerg. Infect. Dis. 7:920-926). VIG was obtained from persons who were immunized with Dryvax. VIG is effective in reducing morbidity and mortality from vaccine related complications, although it has to be given in large amounts and is currently in short supply. Obtaining more of this material requires VV vaccination of people, and that poses the very same risks to the vaccinees who are used as a source of new VIG. Therefore, alternative treatments of complications from VV vaccination are needed in the event that a smallpox outbreak occurs.
In sum, there is a long-felt need to develop a smallpox vaccine that offers advantages over vaccinia virus live vaccines, which have numerous serious drawbacks, and to develop methods of providing protective immunity to this devastating human pathogen. The present invention meets these needs. Further, the present invention provides novel immune reagents that can be developed into a defined and potent form of VIG (called VIG-R).